Cytoprotection by means of phosphotyrosine

ABSTRACT

The present invention relates to a method for protecting biological material from cell damaging factors, a method for producing a pharmaceutical composition for the prophylactic and/or therapy accompanying treatment of radiation therapy patients and/or chemotherapy patients, a cosmetic composition, and a culture medium.

RELATED APPLICATIONS

[0001] This application is a continuation of co-pending internationalapplication PCT/EP02/03403 filed on Mar. 27, 2002 and designating theU.S., which was published in German, and claims priority of Germanpatent application DE 101 17 834.4 filed on Apr. 3, 2001, which isincorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention is concerned with protecting biologicalmaterial from cell-damaging factors.

[0004] 2. Description of the Related Art

[0005] It is known that the integrity of biological material, such asanimal or human cells, tissues or organisms, can be damaged by a largenumber of a very wide variety of factors. These factors, can, forexample, be energy-rich or ionizing radiation, such as radioactiveradiation, X-radiation, cosmic radiation, etc., or ultraviolet light (UVlight). While ionizing radiation is on the one hand one of the noxiousenvironmental factors, on the other hand it can also be employedusefully in medicine. In this connection mention may be made of X-raydiagnostics, nuclear medicine and radiation therapy. This energy-richradiation is characterized by the fact that it has the ability to ionizemolecules.

[0006] Other important cell-damaging factors are chemical agents, forexample, in the form of environmental poisons, but also such chemicalagents which are used in the context of medical treatments astherapeutic agents, for example cytostatic agents. The latter are achemically heterogeneous group of cytotoxic pharmacological substanceswhich prevent, or delay substantially, the division of functionallyactive cells by affecting their metabolism in a variety of ways.Cytostatic agents are principally used in tumor therapy, with thestarting-point for their activity being the fact that the rate at whichtumor cells divide is higher than that of normal cells. Various groupsof cytostatic agents are known: alkylating agents (e.g., cisplatin);antimetabolites (e.g., folic acid antagonists); mitosis inhibitors,antibiotics (e.g., bleomycin); enzymes (e.g., L-asparaginase); andothers.

[0007] These factors can induce damage at all levels of biologicalorganization, thus, reactions at the molecular or macromolecular (e.g.,nucleic acid) level and at the cellular level, or tissue reactions orreactions involving the entire organism are seen in response to theinfluence of these factors.

[0008] Examples of damage which is induced by energy-rich radiation orultraviolet radiation are an alteration of DNA, that is mutagenesis,which can lead to tumor development, and the degeneration, atrophy,fibrosing or necrosis of such tissues which are subjected to a highlevel of radiation.

[0009] Thus, the development of malignant melanoma, for example, ispromoted by the skin being exposed extensively to the sun.

[0010] As was mentioned at the outset, the human organism is confrontedwith particularly high radiation intensities not only in connection witha high level of exposure to sunlight, but also during X-ray diagnosis,when there is an appropriate medical indication, or when radiationtherapy is used in connection with tumor diseases. Radiologists,dentists, traumatologists, technical assistants in radiology, andworkers in X-ray tube factories, are also particularly at risk in thisconnection.

[0011] The abovementioned cytostatic agents, which are to be categorizedas being particularly important as far as causing cell damage isconcerned, cause damages when used in the human body especially becausethe differences in division rate between normal and tumor cells areinsufficient for selectively targeting tumors in a specific manner. Theundesirable side effects or cytostatic agents therefore result, inparticular, from a general inhibition of the regeneration of rapidlyproliferating tissue. Blood cell formation, epithelia of the mucousmembranes, whose inhibition of regeneration leads to gastrointestinaldisturbances, and skin and skin appendages, whose inhibition ofregeneration leads to hair loss, are particularly affected.

SUMMARY OF THE INVENTION

[0012] Against this background, an object of the present invention is toprovide a protection of biological material from cell-damaging factors.

[0013] According to the invention, this object is achieved by usingphosphotyrosine for the said purposes.

[0014] Phosphotyrosine (also termed P-Tyr below) is a modified aminoacid which is derived from the aromatic amino acid tyrosine and in whichthe hydroxyl group of the phenyl of the side chain has beenphosphorylated. Phosphotyrosine is described in a large number ofbiochemistry, molecular biology and protein chemistry textbooks.

[0015] The inventors have surprisingly found that this phosphorylatedamino acid protects biological material from cell-damaging factors.

[0016] In this connection, within the meaning of the invention,phosphotyrosine is understood as being O-phospho-L-tyrosine(L-3-[4-hydroxyphenyl]alanine-4′-phosphate), as well asO-phospho-D-tyrosine (D-3-[4-hydroxyphenyl]alanine-4′-phosphate) andO-phospho-DL-tyrosine (DL-3-[4-hydroxyphenyl]alanine-4′-phospate).

[0017] In this connection, biological material is to be understood asbeing any biological, structured unit such as a cell (in culture or in atissue formation), tissues, organs, organisms, etc.

[0018] Within the meaning of the invention, cell-damaging factors are tobe understood as being influences which have a negative effect on theintegrity and/or viability of biological material, such as ionizingradiation (radioactive radiation, X-radiation, cosmic radiation, etc.),ultraviolet light and chemicals of any type, in particular cytostaticagents (for example alkylating compounds such as cisplatin).

[0019] By the use of phosphotyrosine is to be understood that thismodified amino acid is either brought into contact, in a suitable, forexample, galenic preparation, with biological material, for example,applied to an organism or introduced into this organism, or brought intocontact, in some form in vitro, with biological material, e.g., isolatedcells, tissues or organs. In this connection, the bringing-into-contactwith the phosphotyrosine can take place before, during or after theexposure to cell-damaging factors.

[0020] Within the meaning of the invention, protection of biologicalmaterial means that the damage induced by said cell-damaging factors isreduced or prevented by phosphotyrosine.

[0021] An advantage of the use of phosphotyrosine according to theinvention is to be seen in the fact that phosphotyrosine can be preparedor obtained cost-saving in large quantities. No peptide synthesis orelaborate protein purification is required for this purpose.

[0022] In addition, phosphotyrosine is markedly more resistant todegradation than are peptides or proteins. As a result, a galenicpreparation is also substantially more simple and more stable.

[0023] Due to the small molecular size of the phosphotyrosine itspossible uptake into the cells to be treated is facilitated, with thisresulting in good cytoprotective activity.

[0024] In addition, because of its small size, there is not expected tobe any danger of immunological reactions when phosphotyrosine isintroduced into or applied onto an organism.

[0025] The cytoprotective properties of phosphotyrosine are alsosurprising because so far completely different activities have beenascribed to it. Thus, for example, Shrikant Mishra and Anne W. Hamburgerhave shown that phosphotyrosine inhibits the growth of human breast andkidney carcinoma cells (“O-phospho-L-tyrosine inhibits cellular growthby activating protein tyrosine phosphatases”, Cancer Research 53, pp.557-563, 1993) and that this growth inhibition is effected in a P-Tyrdose-dependent manner (“Exogenous phosphotyrosine modulates epidermalgrowth factor receptor tyrosine phosphorylation”, Carcinogenesis 14, pp.269-273, 1993). This research group demonstrated in the case of twoother tumor cell lines, i.e. a liver carcinoma cell line andarc-transformed NIH 3T3 cells, the ability of phosphotyrosine to inhibitcell growth (“Association of inhibition of all growth byO-phospho-L-tyrosine with decreased phosphorylation”, Cancer Letters102, pp. 65-71, 1996).

[0026] The inventors of the present invention were unable to confirmthis inhibitory effect of exclusively administering phosphotyrosine ontumor cell growth. On the other hand, they were surprisingly able toobserve that, after irradiation with ionizing rays, there is a higherrate of death in tumor cells which have been pretreated withphosphotyrosine than in tumor cells which have not been pretreated.

[0027] It furthermore emerged that, in contrast to the situation intumor cells, normal cells which have been pretreated withphosphotyrosine exhibit a significantly higher survival rate than donormal cells which have not been pretreated with phosphotyrosine bothafter irradiation with ionizing rays and after treatment with cisplatin.

[0028] This selectivity of the cytoprotective property ofphosphotyrosine for normal cells, that is for healthy, non-tumor cells,was not to be expected in the light of the properties of phosphotyrosinewhich were known in the prior art and which were described in acompletely different connection.

[0029] This object underlying the invention is thus achieved completely.

[0030] In a preferred embodiment, the phosphotyrosine is used forprotecting biological material from radiation, preferably ionizingradiation and/or ultraviolet light.

[0031] This has the particular advantage that thereby a use is providedwhich offers protection from particularly important cell-damagingfactors to which virtually every human organism is also at leastpartially exposed.

[0032] Furthermore it is preferred to use phosphotyrosine for protectingbiological material against chemicals, preferably cytostatic agents.

[0033] As mentioned at the outset, chemicals, in particular cytostaticagents, play an important role as cell-damaging factors.

[0034] This preferred use of phosphotyrosine according to the inventionconsequently provides effective protection against particularlyimportant cell-damaging factors.

[0035] In a preferred further development, phosphotyrosine is used forprotecting the skin.

[0036] This has the particular advantage that thereby an organ isprotected which is constantly exposed to cell-damaging factors of aphysical or chemical nature, for example in the form of solar radiationor in the form of environmental poisons.

[0037] In this connection, it is also advantageous that phosphotyrosineis stable under oxidizing conditions, that is, for example, in air, andcan exert a long-lasting protective effect, e.g., against the UV rays ofthe sun. Phosphotyrosine is therefore suitable for being used forprotecting the skin against a high level of solar irradiation. The useof phosphotyrosine for this purpose has the further advantage that,because of its small size, phosphotyrosine can penetrate into the skinand persists in this location for a long period.

[0038] Furthermore it is preferred to use phosphotyrosine within thecontext of a radiation therapy for tumor patients.

[0039] In such a radiation therapy, ionizing radiation is used, forexample, for treating malignant neoplasias. The aim in this connectionis to damage the tumor tissue to the greatest possible extent while atthe same time sparing the surrounding healthy tissue. Because of itspreviously mentioned properties, as identified by the inventors, the useof phosphotyrosine in this connection has the particular advantage thatit selectively protects healthy, that is normal, tissue from cell damagebut does not, on the other hand, display any cytoprotective propertiesfor tumor tissue and, on the contrary, even promotes the death of suchtissue.

[0040] Furthermore, it is preferred to use phosphotyrosine within thecontext of a chemotherapy for tumor patients.

[0041] In chemotherapy, chemotherapeutic agents or cytostatic agents areused specifically for inhibiting the growth of tumor cells in the body,with the aim being to protect healthy cells from the cytotoxicactivities of the chemotherapeutic agents. Because of its propertieswhich have been identified and described above, the use ofphosphotyrosine according to the invention consequently has theparticular advantage of providing a cytoprotector which is selective forhealthy cells.

[0042] An object of the present invention is also the use ofphosphotyrosine for producing a pharmaceutical composition for theprophylactic and/or therapy-accompanying treatment of radiation therapypatients and/or chemotherapy patients.

[0043] For this, the phosphotyrosine can be prepared in the galenicalswhich are in each case appropriate and usual, i.e. be present, whereappropriate, together with usual carrier substances, auxiliarysubstances and/or additives. A pharmaceutical composition of this naturecan be administered intravenously, example percutaneously, by means of alocal injection, for into the regions or cavities of the body which aredirectly affected by the cell-damaging factor, or else by means of beingapplied locally.

[0044] Such a use for producing a pharmaceutical compositionadditionally has the advantage that, as the active substance in thiscomposition, the modified amino acid in question can express itscytoprotective effect without at the same time eliciting any dangerousimmune reactions. Thus, because of its small size, phosphotyrosine onlyhas low immunogenicity, which means that no allergic reaction is to beexpected when the pharmaceutical composition is used on or in the humanor animal body, and that antibodies are not involved in eliminating thephosphotyrosine from the given organism in this connection.

[0045] The cytoprotective activity of phosphotyrosine, which activity isselective for healthy, normal cells, and its toxicity-promotingproperty, which is selective for tumor cells in connection withirradiation, makes the use of this compound particularly suitable forproducing a pharmaceutical composition.

[0046] Another object of the invention is a cosmetic composition whichcomprises phosphotyrosine and, where appropriate, other usual carriersubstances, auxiliary substances and/or additives.

[0047] Such a cosmetic composition can be administered, for example, asa sunscreen milk, skin cream or the like. It then comprises the usualconstituents of such compositions (such as oils, emulsions, pigments,etc.). The cosmetic composition can, of course, additionally comprise UVfilters such as, derivatives of p-amino benzoic acid, salicylic acid,cinnamic acid, dibenzoylmethane or the like.

[0048] As a result of the cytoprotective activity of thephosphotyrosine, such a cosmetic composition provides ideal protection,especially against the UV radiation in sunlight. Since, because of itssmall size, the phosphotyrosine can even penetrate into the skin, andis, furthermore, stable over a long period, it is possible, in this way,to achieve long-term protection against radiation. In this connection,it is also necessary to consider creams which can provide effective skinprotection, for example by the cream being worked into the hands, forindividuals who have a high level of involvement with toxic chemicals.

[0049] Object of the present invention is also a culture medium whichcontains phosphotyrosine and, where appropriate, other usual bufferingsubstances, carrier substances, auxiliary substances and/or additives.

[0050] It has emerged that, when being transported, with this transportfrequently taking place using aircraft at a great height, cells inculture, or else organs, lose 50% or more of their viability as aresult, for example, of being stressed by cosmic radiation. Apart fromproviding suitable conditions for storage and cultivation, the culturemedium according to the invention also provides the biological materialwhich is present in the culture with an effective protection againstcell-damaging factors, e.g., radiation, as a result of thecytoprotective properties of the phosphotyrosine. This thereby ensuresthat cell samples or organs even withstand long routes of transport, forexample by using aircraft, with little or no loss of viability.

[0051] In the culture medium according to the invention it is preferredto use phosphotyrosine in a concentration range of from 1 to 100 μM.Thus, investigations carried out by the inventors have shown that thecytoprotective effect of the phosphotyrosine is particularly high inthis range. This finding is especially surprising because theabovementioned experiments performed by Mishra and Hamburger werecarried out at P-Tyr-concentrations of 1.67 mM, with some even beingcarried out at 16.7 mM, even if these experiments demonstrated adifferent effect of the phosphotyrosine.

[0052] It will be understood that the features mentioned above, andthose which are still to be explained, can be used not only in thecombinations which are in each case indicated but also in othercombinations, or on their own, without departing from the scope of thepresent invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Further advantages are evident from the following embodiments andin connection with the drawings, in which:

[0054]FIG. 1 shows the chemical structural formula of phosphotyrosine(P-Tyr) or O-phospho-L-tyrosine(L-3-[4-hydroxy-phenyl]alanine-4′-phosphate) in ionized form.

[0055]FIG. 2 shows the survival of normal skin fibroblasts, as comparedwith transformed fibroblasts, after pretreatment with differentP-Tyr-concentrations and after radioactive irradiation.

[0056]FIG. 3 shows a comparison of the radioprotective effect orphosphotyrosine, phosphoserine and phosphothreonine on normalfibroblasts.

[0057]FIG. 4 shows the radioprotective effect of a preincubation withP-Tyr on healthy non-tumor cells as compared with tumor cells.

[0058]FIG. 5 shows the clonogenic survival of normal fibroblasts afterirradiation with UVB, in dependence on a pretreatment with P-Tyr, ascompared with tumor cells.

[0059]FIG. 6 shows the clonogenic survival of normal fibroblasts aftertreatment with cisplatin, in dependence on a pretreatment with P-Tyr, ascompared with tumor cells.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT EXAMPLE 1

[0060] Cell cultures used for investigating the cytoprotective effect ofphosphotyrosine

[0061] Human fibroblasts (cell strains HSF1 and HSF6, which are derivedfrom human skin, passages 9 to 15; CCD32, which is derived fromembryonic lung tissue, passages 9 to 11) are particularly well suitedfor investigating the cytoprotective properties of the phosphotyrosine.

[0062] In addition, it is possible to use the human squamous carcinomacell lines HTB-35 (ATCC, which is derived from a cervical carcinoma,passage unknown, used in passage 10 after receipt) (Srivastava et al.,“The status of the p53 gene in human papilloma virus positive ornegative cervical carcinoma cell lines”, Carcinogenesis 13, pages1273-1275, 1992) and HTB-43 (ATCC, is derived from a hypopharyngealtumor, passage 124). Both carcinoma cell lines are characterized by apoint mutation in the p53 gene (Kim et al., “State of p53, Rb and DCCtumor suppressor genes in human oral cancer cell lines”, AnticancerResearch, pages 1405-1413,1993).

[0063] Transformed human fibroblasts, e.g., the cell line HH4dd (isderived from human skin, passages 65 to 70), are also suitable for theinvestigation. This cell line is derived from the normal cell strain HH4and is likewise characterized by a p53 mutation (Dittmann et al., “Theradioprotective effect of BBI is associated with the activation ofDNA-repair relevant genes”, Int. J. Radiat. Biol. 74, pages 225-230,1998). HH4dd grows in soft agar, is characterized by its aneuploidy andinduces tumors in nude mice.

[0064] The cells are cultured in Dulbecco's modified Eagle's medium(DMEM) supplemented with 10% fetal calf serum (FCS) (GIBCO/BRL,Eggenstein, Germany, article No. 40G7285K) (=normal medium), understandard conditions.

EXAMPLE 2

[0065] Irradiating cells, incubating with cisplatin

[0066] The cells, which have grown to confluence, are incubated for 16hours either in simple normal medium or in medium which containsphosphotyrosine, phosphoserine or phosphothreonine (Sigma, Munich,Germany) at the given concentrations.

[0067] For the radioactive irradiation, the cells are irradiated with 4MV photons using a “Linac” (Mevatron⁶⁰/Siemens, Erlangen, Germany) usinga dose rate of 2 Gy/min at room temperature, as described (Dittmann etal. “Bowman-Birk Proteinase inhibitor modulates radiosensitivity andradiation-induced differentiation of human fibroblasts in culture”,Radiother. Oncol. 34, pages 137-143, 1995).

[0068] The cells are irradiated with UV light (312 nm) at a dose rate of450 J/m² per minute using a UVB lamp (Bioblock Scientific, IllkirchCedex, France).

[0069] The incubation with cisplatin take place at a concentration of 1μg of cisplatin/ml (cis-diammine platinum(II-dichloride); Sigma, Munich,Germany).

EXAMPLE 3

[0070] Clonogenic assay (=colony-forming assay)

[0071] The investigations concerning the cytoprotective property ofphosphotyrosine is performed via the so-called clonogenic assay, whichis described, for example, by Dittmann et al., 1995, loc. cit., andwhich is briefly explained below.

[0072] The cells, which are cultured as described in Example 1 and whichare either incubated with the cell-damaging factors as described inExample 2 or remain untreated as controls, are freed from medium, washedand detached from the substrate using 0.05% trypsin and 0.1% EDTA. Forthe purpose of analyzing colony formation, the detached cells are platedat a constant cell density of 1500 cells per 78 cm² dish. The platedcells were then incubated for 14 days in normal medium additionallycontaining 20% FCS. This period of time enabled colony formation to takeplace.

[0073] In this connection, a colony is a mass of cells which develops,during the 14 days of culture, from a single cell as a result ofconsecutive cell divisions. This colony is also termed a clone. In thesense of clonogenic survival, the number of colonies or clonescorresponds to the extent of the damaging effect of a chemical orphysical agent. If many cells die during the treatment with thecell-damaging factors, only a few colonies are then formed after 14days; if many cells survive, many colonies can then be counted after the14 days of culture. Consequently, the clonogenic survival of the cellsafter a cell-damaging treatment is a direct measure of thecytoprotective effect of the phosphotyrosine.

[0074] After the 14 days of culture, the cells are fixed, stained andcounted as described (Dittmann et al., 1995, loc. cit.). Whendetermining clonogenic survival, the colonies containing more than 50cells are counted per plate. The counting take place after the culturedishes have been coded and is carried out by two individualsindependently of each other.

[0075] The result of such a clonogenic assay is expressed as “relativecytoprotection” or in “% number of the clones”, with the number of thecolonies formed in the different assays being related to each other.

EXAMPLE 4

[0076] Reaction of the fibroblasts to radioactive irradiation afterhaving been pretreated with different doses of phosphotyrosine

[0077] Normal skin fibroblasts (HSF6) were incubated for 16 hours withP-Tyr, whose chemical structural formula is shown in FIG. 1, in aconcentration range of from 0 to 2000 μM. The clonogenic survival wasthen determined, as described under Example 3, without any cell-damagingtreatment. The result of such an experiment is shown in FIG. 2A.

[0078] In this figure, the pale grey bars indicate the fraction of theHSF6 cells which survived while the dark grey bars indicate the fractionof the HH4dd cells which survived. As the figure shows, pretreating withP-Tyr alone at concentrations of up to 2000 μM does not have any effecton the clonogenic survival of the HSF6 cells. Comparable results wereobtained for the transformed fibroblast cell line HH4dd. In this case,too, and unexpectedly in view of the results of Mishra and Hamburger(loc. cit.), no effect was seen on clonogenic survival (FIG. 2A).

[0079] On the other hand, the combined treatment with phosphotyrosineand ionizing irradiation, at an energy dose of 4 Gy, demonstrated aclear increase in clonogenic survival in the case of normal fibroblasts.Maximum survival was achieved at a P-Tyr concentration of 10 μM (FIG.2B; pale grey bars). However, with the same treatment, and under thesame exposure conditions, no increase in clonogenic survival wasobserved in the case of the transformed HH4dd fibroblasts. On thecontrary, the treatment of the transformed fibroblasts with 2000 pMP-Tyr resulted in a significant increase in the toxicity of theradiation; see FIG. 2B.

EXAMPLE 5

[0080] Comparison of the radioprotective effect of phosphotyrosine withthat of phosphoserine and phosphothreonine

[0081] In order to test whether other phosphorylated amino acids, suchas phosphoserine (P-Ser) or phosphothreonine (P-Thr), also exhibitradioprotective effects which are comparable with those of P-Tyr,untransformed fibroblasts (HSF1) were pretreated for 16 hours withequimolar concentrations of these two amino acids (in each case 10 μM)and then subjected to an ionizing irradiation with 4 Gy as describedunder Example 2; the results of a clonogenic assay were then comparedwith those for P-Tyr.

[0082] The result of such an experiment is shown in FIG. 3. It is foundthat, while preincubating normal fibroblasts with P-Tyr resulted insignificant radioprotection (2nd bar from the left), incubating withP-Ser or P-Thr under identical experimental conditions did not result inany radioprotection (3rd and 4th bars from the left).

EXAMPLE 6

[0083] Reaction of fibroblasts to different doses of ionizingirradiation after pretreatment with phosphotyrosine.

[0084] The cell strains HSF6 and CCD32, and the cell lines HTB-35 andHTB-43, were preincubated for 16 hours with 10 μM P-Tyr. The cells werethen irradiated with doses of from 0 to 6 Gy and the clonogenic survivalwas determined after a period of 6 hours. Each data point in such anexperiment, shown in FIG. 4A, represents the mean value of severalmeasurements and the standard deviation. The curve fit was calculatedhere using the linear quadratic model and α and β values were determinedand tested for significance using the Student t-test. The asterisksindicate a significance difference (p<0.05) for α or β or both. FIG. 4Bshows, in tabular form, the mean value of the relative cytoprotectionfrom the four measured values (SF4) for each assay and the standarddeviation, including the p-value.

[0085] Pretreatment of normal skin fibroblasts (HSF6) and normal lungfibroblasts (CCD32) with 10 μM P-Tyr lead to a significant increase inclonogenic survival up to a dose of 6 Gy (FIG. 4A, upper row, FIG. 4B,rows 1 and 2). On the other hand, pretreating the transformed cell lines(HTB-35 and HTB-43) with P-Tyr resulted in a significant reduction inclonogenic survival (FIG. 4A, lower row, FIG. 4B, rows 3 and 4).

[0086] These results show that, while a pretreatment of healthy, that isnormal, cells with phosphotyrosine efficiently protects these cells fromcell-damaging factors, a pretreatment of tumor cells withphosphotyrosine leads to an increase in the death of these transformedcells when they are incubated with cell-damaging factors.

EXAMPLE 7

[0087] Reaction of phosphotyrosine-pretreated fibroblasts to UVBirradiation

[0088] In order to test the cytoprotective protection ofphospho-tyrosine in regard to non-ionizing radiation, normal,untransformed, fibroblasts (HSF1) and transformed fibroblasts (HH4dd)were pretreated with 10 μM P-Tyr for 16 hours and then irradiated with200 J of UVB; they are then investigated 7 hours later in a clonogenicassay as described in Example 3.

[0089] In this experiment, it is found that the clonogenic survival ofUVB-irradiated normal, untransformed fibroblasts (HSF1) is increased by37% when they have been pretreated with phosphotyrosine (see FIG. 5A,bars 2 and 3 from the left).

[0090] However, pretreatment with P-Tyr in connection with UVBirradiation had no effect on the clonogenic survival of transformedfibroblasts (HH4dd) (see FIG. 5B, bars 2 and 3 from the left).

[0091] In both cases, unirradiated cells serve as controls (Co), withthe number of clones formed in this case representing 100%.

[0092] This experiment demonstrates that, in the case of non-neoplasticcells, phosphotyrosine also has cytoprotective properties in regard tononionizing radiation. Also in this case, the cytoprotective activity isselective for normal, untransformed cells. Thus, pretreatment with P-Tyrdoes not have any effect on the survival of transformed cells afterirradiation with UVB.

EXAMPLE 8

[0093] Reaction of Fibroblasts to cisplatin treatment after pretreatmentwith phosphotyrosine

[0094] In order to investigate the cytoprotective effect ofphosphotyrosine with regard to chemical cell-damaging factors, forexample cytostatic agents, normal fibroblasts (ESF1) and transformedfibroblasts (HH4dd) were pretreated for 16 hours with 10 μM P-Tyr andthen, after that, incubated for one hour with 1 μg of cisplatin/ml. Thecells were then washed twice with normal medium and plated 6 hourslater. After that, a clonogenic assay was carried out as described underExample 3.

[0095]FIG. 6 shows the result of such an experiment. Pretreatment ofnormal, untransformed fibroblasts (HSF1) with P-Tyr leads, when thesefibroblasts were incubated with cisplatin, to an increase in clonogenicsurvival of 38.7% as compared with the clonogenic survival oftransformed fibroblasts (HH4dd) which have not been pretreated (see FIG.6A, 2nd and 3rd bars from the left).

[0096] However, a pretreatment of transformed fibroblasts (HH4dd) withP-Tyr did not lead to any increase in clonogenic survival in connectionwith cisplatin treatment (FIG. 6B, 2nd and 3rd bars from the left).

[0097] In controls both cases, unirradiated cells once again serve as(Co), with the number of clones formed in this case representing 100%.

[0098] Consequently, phosphotyrosine provides cytoprotection, which isselective for healthy, that is normal, cells, in regard to acell-damaging factor of chemical nature as well.

What is claimed is:
 1. A method of using phosphotyrosine for protectingbiological material from cell-damaging factors, comprising the steps of:(a) providing of biological material, and (b) contacting said biologicalmaterial to phosphotyrosine, said contacting taking place before, duringor after exposure to said cell-damaging factors.
 2. The method of claim1, wherein said cell-damaging factors comprise radiation, and/orultraviolet light.
 3. The method of claim 2, wherein said radiation isionizing radiation.
 4. The method of claim 1, wherein said cell-damagingfactors comprise chemicals.
 5. The method of claim 4, wherein saidchemicals are cytostatic agents.
 6. The method of claim 1, wherein saidbiological material is skin.
 7. The method of claim 1, wherein saidbiological material is a tumor patient subjected to a radiation therapy.8. The method of claim 1, wherein said biological material is a tumorpatient subjected to a chemotherapy.
 9. A method for producing apharmaceutical composition for the prophylactic and/ortherapy-accompanying treatment of radiation therapy patients and/orchemotherapy patients, comprising the steps of: (a) providing ofphosphotyrosine, and (b) formulating said phosphotyrosine into anappropriate galenics.
 10. A pharmaceutical composition, comprisingphosphotyrosine and a pharmaceutically acceptable carrier.
 11. A culturemedium, comprising phosphotyrosine and other usual buffering substances,carrier substances, auxiliary substances and/or additives.
 12. Theculture medium of claim 11, comprising phosphotyrosine in aconcentration range of from about 1 μM to about 100 μM.
 13. A method forprotecting biological material from cell-damaging factors, comprising:(a) providing of biological material, and (b) contacting said biologicalmaterial to phosphotyrosine, said contacting taking place before, duringor after exposure to said cell-damaging factors, said cell damagingfactors being selected from the group consisting of: radiation,ultraviolet light, and chemicals.
 14. The method of claim 13, whereinsaid radiation is ionizing radiation, and wherein said chemicals arecytostatic agents.
 15. The method of claim 13, wherein said biologicalmaterial is skin.
 16. The method of claim 13, wherein said biologicalmaterial is a tumor patient subjected to a radiation therapy.
 17. Themethod of claim 13, wherein said biological material is a tumor patientsubjected to a chemotherapy.
 18. A method for producing a pharmaceuticalcomposition for protection of healthy cells within the frame ofprophylactic and/or therapy-accompanying treatment of radiation therapypatients and/or chemotherapy patients, comprising the steps of: (a)providing a phosphotyrosine, and (b) formulating said phosphotyrosineinto an appropriate galenics.
 19. A culture medium, comprisingphosphotyrosine and other usual buffering substances, carriersubstances, auxiliary substances, and/or additives, wherein said mediumcomprises phosphotyrosine in a concentration range of from about 1 μM toabout 100 μM.
 20. A method for protecting biological material fromcell-damaging factors, comprising: (a) providing of biological material,and (b) contacting said biological material to phosphotyrosine.